This Program Project Grant began 20 years ago when we defined the LDL receptor pathway for the control of cholesterol metabolism and showed that defects in this pathway produce Familial Hypercholesterolemia and its attendant atherosclerosis. After 20 years, our goals remain the same: to understand the genetic and metabolic defects that produce hypercholesterolemia and accelerated atherosclerosis and to use this knowledge to prevent and treat the disease. During the last grant period (Years 16- 19), we have made considerable progress through the discovery that sterols regulate gene transcription by controlling the proteolysis of a membrane- bound transcription factor called SREBP. This process, in turn, controls the level of cholesterol in cells and in blood. Our work has also taken us into related areas of research, including receptor-mediated endocytosis and caveolae-mediated potocytosis, mevalonate metabolism and protein prenylation, the relation between bile acid and cholesterol metabolism, and structure/function studies of the LDL receptor gene family. We now apply for a 5-year renewal of our Program Project Grant (Years 21-25) that will allow us to further study these phenomena through an integrated, multidisciplinary approach. We propose to focus on key molecules involved in: 1) cholesterol homeostasis (LDL receptor, HMG CoA synthase and reductase, SREBP-1 and -2, oxysterol binding protein, cholesterol 7alpha-hydroxylase, oxysterol 7alpha-hydroxylase, sterol 27-hydroxylase); 2) lipoprotein transport (LDL receptor, LRP, VLDL receptor, RAP); 3) regulation of cell growth (prenylated Ras proteins, farnesyltransferase, caveolin): and 4) regulatIon of membrane traffic (prenylated Rab proteins, Rab geranylgeranyl transferase, REPs). A series of model systems will be used to study the mechanisms by which these proteins operate at the molecular level (i.e., the gene, the mRNA, and the protein), at the level of the intact cell, and at the level of the whole animal. In conducting these studies, we will use a wide variety of techniques, including biochemistry, immunology, molecular biology, genetics, cell biology, electron microscopy, transgenic and knockout mice, animal physiology, and clinical genetics. The successful completion of these studies should enhance understanding of the molecular basis of hypercholesterolemia and atherosclerosis and also provide insights into the ways in which lipid-modified proteins regulate cell proliferation and membrane traffic.